Transcript Transformation Lab - Central Magnet School

Mrs. Stewart
Medical Interventions
Central Magnet School
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We will demonstrate the ability to transform
bacterial cells with free-floating DNA using a
gene that codes for Green Fluorescent
Protein (GFP).
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The genetic
modification of a
bacterium by the
incorporation of
free-floating
DNA from the
surrounding
environment
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Gene found in bioluminescent
jellyfish Aequorea victoria
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Gene causes jellyfish to
fluoresce and glow in the dark
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If transformation is successful,
bacteria will produce this
protein and fluoresce under UV
light
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Ori = origin of replication
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araC = arabinose detection
gene that regulates the
production of GFP
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Bla = beta – lactamase
enzyme gene (antibiotic
resistance to beta-lactam
abx)
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GFP = green fluorescent
protein gene inserted into
plasmid using restriction
enzymes
Gene expression in all organisms is carefully regulated to allow for adaptation to
differing conditions and to prevent wasteful overproduction of unneeded
proteins. The genes involved in the breakdown of different food sources are
good examples of highly regulated genes. For example, the simple sugar
arabinose is both a source of energy and a source of carbon for bacteria. The
bacterial genes that make digestive enzymes to break down arabinose for food
are not expressed when arabinose is not in the environment. But when
arabinose is present, these genes are turned on. When the arabinose runs out,
the genes are turned off again.
Arabinose initiates transcription of these genes by promoting the binding of RNA
polymerase. In the genetically engineered pGLO plasmid DNA, some of the
genes involved in the breakdown of arabinose have been replaced by the
jellyfish gene that codes for GFP. When bacteria that have been transformed
with pGLO plasmid DNA are grown in the presence of arabinose, the GFP gene
is turned on and the bacteria glow brilliant green when exposed to UV light.
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Student Stations
 LB agar plate
 Alcohol burner
 Permanent marker
 Inoculation loop
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Common Materials
 E. coli
 Transformation solution
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The purpose of
this starter plate
is to have
SINGLE
COLONIES
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Streak for
colonies
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Student Stations
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Group E. coli starter plate
2 – 1.5 ml micro test tubes + tube rack
4 - agar plates – LB, 2 LB/amp, LB/amp/ara
Transformation solution
LB broth
Inoculation loops
Micropipet (.5-10 µl) + tips
3 - Sterile disposable pipets
Permanent marker
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Label the 2 micro test tubes
 “+ pGlo” on one tube
 “- pGlo” on one tube
 Add group name to both
 Place tubes in tube rack
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Transfer 250 µl of
transformation solution
(CaCl2)into each tube*
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Place tubes on ice to
maintain sterility
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*Purpose: Both DNA and a bacterial
cell wall have a negative charge.
CaCl2 will help neutralize those
opposing charges to increase change
of DNA uptake
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Use a sterile loop to pick up 3 or 4
LARGE colonies with a uniformly
circular shape and smooth edges.
 Do not use a swab of bacteria from the dense
growth – we need actively growing bacteria for
optimum transformation
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Transfer loop w/ bacteria into
+pGlo tube and spin until colony is
dispersed.
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Repeat for –pGlo tube
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Examine the pGlo DNA
solution with the UV light
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Record observations
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Transfer 10 µl pGlo DNA
solution into +pGlo tube
 DO NOT add pGlo plasmid DNA to –pGlo tube
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Incubate on ice for 10 minutes
10 minutes
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Label the 4 agar plates as follows:
 Add group name to all 4 of them
 Label 2 plates +pGlo - LB/amp and LB/amp/ara
 Label 2 plates –pGlo – LB/amp and LB
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Transfer both tubes from
ice to heat block (42oC) for
exactly 50 seconds
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Immediately transfer back
to ice after the 50 seconds
has ended
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Incubate on ice for 2
minutes
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Remove tubes from ice
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Add 250 µl of LB nutrient
broth to each tube using a
sterile pipet
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Incubate at room temp for
10 minutes
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Gently flick/tap
tubes with your
finger to mix and
resuspend bacteria
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Pipet 100 µl of
transformation of
each tube onto the
corresponding
plates
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Use a new sterile loop for each plate
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Spread the suspensions evenly around the
plate surface
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DO NOT puncture the agar by pressing too
deep
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Stack plates and tape
together
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Incubate
 3rd period – countertop
 2nd period – incubator
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Store them upside down!